Central sensitization is the hyperexcitability of spinal processing after peripheral nerve injury or inflammation. This phenomenon may be associated with nitric oxide (NO) signal pathway in synapse. Here, we have investigated the effect of NO on hyperpolarization-activated inward current (I(h)) in substantia gelatinosa (SG) neurons, using the whole-cell patch clamp technique. I(h) was increased by the application of sodium nitro prusside (SNP, a NO donor) or 8Br-cGMP. The stimulatory effects of NO were abolished by guanylyl cyclase inhibitor, ODQ, suggesting that the effect of NO was mediated by cGMP. However, this effect of NO was not prevented by the pretreatment with KT5823, PKG inhibitor. Taken together, the activation of I(h) in SG neurons could be mediated by NO-cGMP dependent pathway. These results reveal an involvement of NO in excitability of SG neuron via the activation of I(h) may be associated with central sensitization.